利用共转化法获得无标记转WYMV-Nib8基因小麦
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摘要
随着转基因技术的广泛应用及转基因作物的逐步商业化，转基因作物的生物安全性，尤其是抗生素或除草剂抗性的选择标记基因存留于转基因植物中引起的安全性问题日益成为人们关注的焦点。小麦是世界上栽培面积最大的粮食作物之一，目前还没有转基因小麦的商品化推广，剔除转基因小麦中选择标记基因，从而消除了转基因作物中选择标记基因带来的生物安全性问题，对于转基因小麦的进一步商品化具有十分重要的意义。因此，通过一些策略获得无标记的转基因植株成为转基因科研工作者研究的主要目标之一。
1．本研究在所在实验室先期已将小麦黄花叶病毒(Wheat Yellow Mosaic Virus,WYMV)复制酶基因Nib8(构建在表达载体pUbiNib8上)和含有筛选标记bar基因(构建在表达载体pAHC20上)利用基因枪法共转化导入扬麦158小麦的基础上，在转基因抗病株系N12小麦的T_3代中，采用PCR分子检测技术，从检测的268株中筛选得到28株只含功能基因(WYMV-Nib8)而没有选择标记基因bar的扬麦158转基因植株。
2．本研究在268株转基因小麦PCR分子检测结果的基础上，进一步证实转基因小麦中的外源基因可通过配子从T_2代传递到T_3代，并分析了外源基因在T_3代转基因小麦中的遗传分离规律，其大多数趋向于1：1的非孟德尔遗传。可能是发生了转基因丢失、截短、重排等因素，有待于进一步研究。相信随着转基因研究水平的提高和转化技术的进步，研究者们将越来越明了转基因在后代中的遗传传递规律。
3．经PCR分子检测基因型为Nib~+bar~-的20个株系播种在江苏姜堰、仪征两个试验点进行抗病性功能鉴定，除草剂抗性鉴定在中国农科院试验田进行，抗病性及除草剂抗性鉴定结果与PCR分子检测结果基本相符。基因型为Nib~+bar~-的转基因小麦能耐受的Basta浓度为20ppm。鉴定结果表明，我们获得了19个无选择标记基因bar、带WYMV复制酶基因Nib8的小麦黄花叶病抗性稳定遗传的转基因株系，为抗小麦黄花叶病的转基因小麦的环境释放、安全性评价及进一步商品化打下了良好的基础。
利用共转化法剔除选择标记基因，获得有功能的转基因小麦目前在国内尚无成功的报道。
With the development of plant biotechnology, genetic transformation of crop plants is becoming increasingly routine and shows its potential application value in crop breeding. Transgenic crops have been developed in commercialized scale in some countries,but their biosafety have attracted public concerns in the world at the same time .Therefore,it is becoming more and more important to eliminate the selective genes because of their negative function, such as antibiotics resistance and herbicide resistance .Furthermore , marker-free transgenic plants(MFTPs) have a number of special advantages ,such as decreasing the conerns about safety of selective gene and stack of transgenes progressively into transgenic plants.Major approachs used recently for obtaining MFTPs were reviewed in this paper,which has shown a tendency to deleting any unuseful DNA sequences in the final product.
(1) In this study, 268 lines of TS transgenic wheat from T2 generation line N12 of Yangmai 158 containing Wheat Yellow Mosaic Virus Nib8 gene(WYMV-Nib8) and bar selection gene that were obtained by Co-transformation of biolistic particle were analysized by PCR procedure .The results indicated that 28 lines only with the target gene were identified finally.
(2) Genetic segregation of interest gene and bar gene in the Ta generation were studied in the paper,and segregation ratio was 1:1 approximately.Our results showed that foreign DNA integrated were inherited in aberrant Mendelian manner.The reasons may be loss, truncation and rearrangement of transgene and so on.With the development of study of offspring of transgenic plants and technique of transformation,genetic regulation of transgene in plants will be identified.
(3) The resistance to WYMV of 20 lines whose genotype were Nib+bar- by PCR analysis were tested in Jiangyan , Yizheng citiesJiangSu province,and the resistance to herbicide were tested in CAAS.The results of resistance testing agreed with PCR analysis roughly.lt was proven that 19 out of 20 lines contained WYMV-Nib8 gene without selective marker gene (bar) and the resistance to WYMV could be inherited stablly .The obtainment of 19 transgenic lines made a full preparation for releasing in environment , evaluating biosafety and commercilization of transgenic wheat.
It hasn't been reported at present in China that transgenic wheat conferring resistance to WYMV without bar gene were obtained by Co-transformation.
1．本研究在所在实验室先期已将小麦黄花叶病毒(Wheat Yellow Mosaic Virus,WYMV)复制酶基因Nib8(构建在表达载体pUbiNib8上)和含有筛选标记bar基因(构建在表达载体pAHC20上)利用基因枪法共转化导入扬麦158小麦的基础上，在转基因抗病株系N12小麦的T_3代中，采用PCR分子检测技术，从检测的268株中筛选得到28株只含功能基因(WYMV-Nib8)而没有选择标记基因bar的扬麦158转基因植株。
2．本研究在268株转基因小麦PCR分子检测结果的基础上，进一步证实转基因小麦中的外源基因可通过配子从T_2代传递到T_3代，并分析了外源基因在T_3代转基因小麦中的遗传分离规律，其大多数趋向于1：1的非孟德尔遗传。可能是发生了转基因丢失、截短、重排等因素，有待于进一步研究。相信随着转基因研究水平的提高和转化技术的进步，研究者们将越来越明了转基因在后代中的遗传传递规律。
3．经PCR分子检测基因型为Nib~+bar~-的20个株系播种在江苏姜堰、仪征两个试验点进行抗病性功能鉴定，除草剂抗性鉴定在中国农科院试验田进行，抗病性及除草剂抗性鉴定结果与PCR分子检测结果基本相符。基因型为Nib~+bar~-的转基因小麦能耐受的Basta浓度为20ppm。鉴定结果表明，我们获得了19个无选择标记基因bar、带WYMV复制酶基因Nib8的小麦黄花叶病抗性稳定遗传的转基因株系，为抗小麦黄花叶病的转基因小麦的环境释放、安全性评价及进一步商品化打下了良好的基础。
利用共转化法剔除选择标记基因，获得有功能的转基因小麦目前在国内尚无成功的报道。
With the development of plant biotechnology, genetic transformation of crop plants is becoming increasingly routine and shows its potential application value in crop breeding. Transgenic crops have been developed in commercialized scale in some countries,but their biosafety have attracted public concerns in the world at the same time .Therefore,it is becoming more and more important to eliminate the selective genes because of their negative function, such as antibiotics resistance and herbicide resistance .Furthermore , marker-free transgenic plants(MFTPs) have a number of special advantages ,such as decreasing the conerns about safety of selective gene and stack of transgenes progressively into transgenic plants.Major approachs used recently for obtaining MFTPs were reviewed in this paper,which has shown a tendency to deleting any unuseful DNA sequences in the final product.
(1) In this study, 268 lines of TS transgenic wheat from T2 generation line N12 of Yangmai 158 containing Wheat Yellow Mosaic Virus Nib8 gene(WYMV-Nib8) and bar selection gene that were obtained by Co-transformation of biolistic particle were analysized by PCR procedure .The results indicated that 28 lines only with the target gene were identified finally.
(2) Genetic segregation of interest gene and bar gene in the Ta generation were studied in the paper,and segregation ratio was 1:1 approximately.Our results showed that foreign DNA integrated were inherited in aberrant Mendelian manner.The reasons may be loss, truncation and rearrangement of transgene and so on.With the development of study of offspring of transgenic plants and technique of transformation,genetic regulation of transgene in plants will be identified.
(3) The resistance to WYMV of 20 lines whose genotype were Nib+bar- by PCR analysis were tested in Jiangyan , Yizheng citiesJiangSu province,and the resistance to herbicide were tested in CAAS.The results of resistance testing agreed with PCR analysis roughly.lt was proven that 19 out of 20 lines contained WYMV-Nib8 gene without selective marker gene (bar) and the resistance to WYMV could be inherited stablly .The obtainment of 19 transgenic lines made a full preparation for releasing in environment , evaluating biosafety and commercilization of transgenic wheat.
It hasn't been reported at present in China that transgenic wheat conferring resistance to WYMV without bar gene were obtained by Co-transformation.
引文
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